Our studies have defined signaling cascades regulating Cryptococcus neoformans differentiation and virulence. As one paradigmatic example, the Ca2+ activated phosphatase calcineurin was demonstrated to govern a signaling cascade enabling growth at mammalian body temperature and virulence. In parallel, calcineurin was found to be required for mating and monokaryotic fruiting, differentiation cascades thought to produce the suspected infectious propagules in nature. Our efforts supported by this proposal have resulted in elucidating many pathway components, including the Ca2+ sensor calmodulin, the calcineurin catalytic and regulatory subunits, the conserved calcineurin binding protein and pathway effector Cbp1, the immunophilins cyclophilin A and FKBP12, and the cell wall regulatory component Cts1. In summary, we have achieved the goals of the two previous awards, and contributed a wealth of additional methodological and conceptional advances fueled by this support. As one example, we recently reported that monokaryotic fruiting represents a modified sexual cycle involving partners of the same mating type. These advances enable us to broaden the scope of this competitive renewal from an original focus on calcineurin in mating and fruiting to a genetic and genomic analysis of mating and fruiting in C. neoformans. Previous studies have linked mating type to virulence of C. neoformans. A central conundrum has been the finding that the vast majority of clinical and environmental isolates are of the mating type. Our discovery of same sex mating provides an explanation how diversity can be maintained in a largely unisexual population. Here, we propose to analyze in detail the monokaryotic fruiting sexual cycle and its impact on the organism. We propose three inter-related specific aims. Aim 1 is a physiological, genetic, and genomic analysis of monokaryotic fruiting including whole genome microarray, insertional mutagenesis, and quantitative trait mapping. In aim 2, we will dissect the role of the Cpr2 pheromone receptor homolog in monokaryotic fruiting. Aim 3 is to examine the role of same-sex mating in the origin of the C. gattii Vancouver Island outbreak. These studies will contribute to an understanding of the roles of sexual reproduction in microbial pathogenesis, and provide insights into the life cycle and virulence cycle of C. neoformans and C. gattii applicable to development of treatment approaches, diagnostics, or environmental interventions to reduce exposure to infectious spores.